The ionophore X-537A has been shown to induce profound physiological changes in cardiac activity characteristic of positive inotropism, but the biochemical basis of these effects remains poorly understood. Characterization of the complexes formed between X-537A and mono- and divalent cations, and catecholamines in membranes phospholipid bilayer vesicles should provide information essential for design of a therapeutic application in cases of cardiac insufficiency. We hope to characterize the thermodynamic properties of inorganic cation complexes of the ionophore in the vesicle system, employing techniques which we have already developed for bulk organic solvents of various polarities. We will use flurorescence and circular dichroism as measures of complex formation to determine stoichoimetries and equilibrium constants in vesicles. Kinetic studies will be continued in bulk solvents, employing nuclear magnetic resonance to calculate exchange rates, and stopped-flow fluorescence spectroscopy to monitor binding and substitution reactions. The rotational motion of X-537A in membranes will be studied by fluorescence depolarization, making use of the marked temperature dependence of fluidity in bilayers of different homogeneous phospholipid compositions. Such temperature effects can be correlated with electron spin resonance measurements of spin labeled lipid probes which may be simultaneously introduced into the bilayer. We intend to pursue our initial observations of fusogenic properties of X-537A by observing its effects on membrane structure. Electron microscopy of negatively stained preparations will provide a direct measure of increases in vesicle size induced by X-537A. Effects of the ionophore on membrane fluidity may be observed with ESR probes, and kinetics of membrane fusion may be followed by light scattering.